Electrophoretic Technique: Capillary Zone Electrophoresis

Chapter 16

Electrophoretic Technique: Capillary Zone Electrophoresis Gerardo A´lvarez-Rivera*, Alejandro Cifuentes* and Marı´a Castro Puyana† *

Foodomics Laboratory, Institute of Food Science Research (CIAL, CSIC), Madrid, Spain, Department of Analytical Chemistry, Physical Chemistry and Chemical Engineering, University of Alcala´, Madrid, Spain †

Chapter Outline 1 Introduction 2 Equipment and Instrumentation Used in CE 3 Theory and Principles of CE 4 Modes of CE 4.1 Free-Solution Capillary Electrophoresis 4.2 Micellar Electrokinetic Chromatography 4.3 Capillary Gel Electrophoresis 4.4 Capillary Isoelectrofocusing 4.5 Capillary Electrochromatography

1

659 663 664 667 667 667 668 669

5 Application of CE to Food Authentication 670 5.1 DNA Analysis 670 5.2 Analysis of Proteins 673 5.3 Analysis of Chiral Compounds 674 5.4 Analysis of Other Compounds 677 5.5 CE Microchip Technology in Food Authentication 680 6 Future Outlooks 680 7 Conclusions 681 Acknowledgments 681 References 681

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INTRODUCTION

Food authenticity is crucial in order to assure food safety and quality, including health concerns, such as, for example, allergies to proteins nondeclared in the label, besides other evident economic and religious consequences. These implications have made that consumers, producers, and regulators demand faster and more powerful analytical procedures to guarantee authenticity of foods. This demand has brought about the rapid scientific and technological development of new methods and techniques that allow the determination of food authenticity. A good example of the multiple food authenticity issues that nowadays can be addressed is provided in Table 1. Thus, common adulteration practices Modern Techniques for Food Authentication. https://doi.org/10.1016/B978-0-12-814264-6.00016-5 © 2018 Elsevier Inc. All rights reserved.

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TABLE 1 Examples of Food Authenticity Issues Commodity

Issue

Reference

Beverages

Authenticity of seized whiskey adulterated by dilution with tap water

Rezende et al. (2016)

Detection of blends of high-value juices (orange and pineapple) with cheaper alternatives

Navarro-Pascual-Ahuir et al. (2015)

Classification of wine for authentication assurance

Gomez and Silva (2016), Zeravik et al. (2016)

Distinction between different Irish whiskies according to the aging process and mashbill

White et al. (2017)

Addition of racemic α-hydroxy acids in wine

Kamencev et al. (2016)

Detection of GM maize lines

Holck and Pedersen (2011)

Detection of GM raise events

Mingzhe et al. (2016)

Assurance of barley malts quality from diverse geographical region.

Kaur et al. (2015)

Identification of GM

Basak et al. (2014)

Authentication of fruit products according to their origin

Navarro et al. (2014)

Addition of racemic α-hydroxy acids in fruits juices

Kodama et al. (2010)

Provide the missing information on the nutritional value of saffron

Hashemi and Erim (2016)

Authentication of mentha and peppermint teas

Roblova et al. (2016)

Pork meat adulteration in “halal” meat

Barakat et al. (2014)

Traceability and seafood origin verification

El Sheikha and Montet (2016)

Perch authentication according to the geographical localization

Rolli et al. (2014)

Determination of milk adulteration by whey addition

de Oliveira Mendes et al. (2016)

Distinction between camel milk and bovine milk

Omar et al. (2016)

Adulteration of ewe milk with cow milk

Trimboli et al. (2017)

Cereals

Fruit and vegetable

Herbs and spices

Meat and fish

Milk and dairy products

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TABLE 1 Examples of Food Authenticity Issues—cont’d Commodity

Issue

Reference

Oils and fats

Cultivar origin authentication of monovarietal olive oils

Uncu et al. (2015)

Detection of undeclared addition of other olive oils to monovarietal extra virgin olive oil

Bazakos et al. (2016)

Undeclared food allergen contents on the label

Cheng et al. (2015) and Maria Lopez-Calleja et al. (2017)

Characterization of monofloral honeys from different botanical origins

Kaygusuz et al. (2016)

Authentication of green coffee beans (Arabica & Robusta)

Spaniolas et al. (2014)

Maple syrup adulteration with less expensive sugars

Taga and Kodama (2012)

Distinction between coffee seeds treated with different fermentation processes

Vaughan et al. (2016)

Others

include the fraudulent substitution of more expensive animal and plant species with cheaper ones; the addition of nonanimal proteins to meat products; the presence of protected or nonauthorized organisms (species, genetically modified varieties) in foods, etc. Although these are relevant issues to both food industry and regulatory agencies, food authenticity (including identification of animals or plants species, geographical origin, processing, etc.) is a difficult task. For this reason, the verification of food authenticity has to be faced by developing selective and sensitive methods able to find a compound (or group of compounds) that can be used as marker of a given species, geographical origin, food processing, etc. (Sotelo and P
erez-Martı´n, 2003). Therefore, faster, more powerful, cleaner, and cheaper analytical procedures are being required by food chemists, regulatory agencies, and quality control laboratories to meet these demands. As a consequence, there is a growing interest in the development of innovative analytical procedures meeting all the requirements aforementioned for ensuring the authenticity of the food supply. Among the different analytical techniques that can be employed to analyze foods and food compounds, the use of capillary electrophoresis (CE) has emerged as a good alternative due to the multiple analytical advantages that this technique provides. Thus, CE offers high analysis speed, high separation efficiencies, great variety of applications, reduced sample and solvents

662 Modern Techniques for Food Authentication

consumption, and automatization. These characteristics have contributed to the increasingly growing number of applications of CE in food science and technology (Cifuentes, 2006; Acunha et al., 2016a) since its development in the 1980s (Jorgenson and Lukacs, 1983). A wide array of molecules including small ions, amino acids, carbohydrates, organic acids, vitamins, lipids, flavonoids, additives, contaminants, peptides, proteins, and DNA fragments have been analyzed using CE in different food matrices (Cifuentes, 2006; Acunha et al., 2016a). As an example of the large interest that the use of CE in food analysis has brought about, Table 2 shows

TABLE 2 Reviews on Capillary Electromigration Methods in Food Analysis and Related Areas Subject

Publication Year

Reference

CE in meat adulteration

2017

Iammarino et al. (2017)

CE in food analysis

2008–2018

Acunha et al. (2016a), A´lvarez et al. (2018), Castro-Puyana et al. (2012), Garcı´a-Can˜as and Cifuentes (2008), Garcı´a-Can˜as et al. (2014), and Herrero et al. (2010)

CE of nonprotein amino acids as food quality markers

2016

Perez-Miguez et al. (2016)

Capillary electrochromatography in food analysis

2016

D’Orazio et al. (2016)

Open tubular-capillary electrochromatography

2016

Tarongoy et al. (2016)

Amino acid analysis by CE methods

2016

Poinsot et al. (2016)

CE with chemiluminescence detection in food analysis

2016

Lara et al. (2016)

Simultaneous CE separation of cations and anions

2016

Koenka et al. (2016)

Microchip electrophoresis for wine analysis

2016

Gomez and Silva (2016)

CE of amino acids, proteins, carbohydrates and lipids in food

2016

de Oliveira et al. (2016)

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TABLE 2 Reviews on Capillary Electromigration Methods in Food Analysis and Related Areas—cont’d Publication Year

Reference

CE of pesticides in environmental and food samples

2016

Bol’shakova and Amelin (2016)

Chiral separations using miniaturized techniques

2016

Fanali and Fanali (2016)

Coupled solid-phase extraction-capillary electrophoresis

2016

Ramautar et al. (2016)

Capillary electrophoresis with mass spectrometry

2015

Klepa´rnı´k (2015)

Capillary and microchip analysis of PAHs

2015

Ferey and Delaunay (2015)

Subject

some useful reviews mostly published in the period 2015–2018 on this topic; readers interested in getting more information on this subject can resort to these reviews. In the following sections, a description of the basic instrumentation, operating principles, and modes of CE is provided. Next, a revision of the main applications of this analytical technique in food science and technology is given, with special focus on its relevance for food authentication. This chapter concludes with a critical discussion about the main advantages and disadvantages of CE and some future outlooks of this technique in the food analysis domain.

2

EQUIPMENT AND INSTRUMENTATION USED IN CE

A scheme of the basic instrumentation required in CE equipment is shown in Fig. 1. The separation of the analytes is performed inside the capillary, which is usually made of fused silica. The capillary dimensions range from 25 to 100 μm of inner diameter and from 25 to 100 cm of length. These capillaries of silica are fragile so they are externally coated with polyimide for the addition of flexibility and resistance. The buffer-filled capillary is placed between two vials usually filled with the same buffer. During the injection, the inlet buffer is substituted by a vial containing the sample. A small volume of sample (nanoliters) is introduced in the capillary by pressure, vacuum, or applying a difference of voltage (electromigration). Once the injection is done, the vial containing the sample is changed

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Detector Electrode A Capillary Buffer vial

HV power supply

Buffer vial

Sample vial

FIG. 1 Basic capillary electrophoresis instrumentation.

by the vial with the separation buffer. After that, an electric field is applied to start the separation. In CE equipment, the high voltage power supplies employed usually provide voltages ranging from 0 to 30 kV. The analytes are separated based on their different electrophoretic mobility under the influence of the electrical field moving to the detection point as pure bands. Moreover, the capillary needs to be thermostatized, dissipating in this way the heat generated by Joule effect and maintaining a constant temperature from one analysis to another, assuring reproducibility. A small section of the outer polyimide coating is removed near the outlet end of the capillary to form the detection window, therefore, the detection is done on-column (i.e., in the same capillary). This type of continuous detection has permitted the automatization of this technique, and has eliminated dead volumes by avoiding any connection what increases separation efficiency. Besides, the technique allows carrying out quantitative analysis. On the other hand, the narrow optical pathlength of these detection windows (25–100 μm) and the low injection volumes (nanoliters) provide poor detection limits, making it difficult for the application of CE for traces analysis. Fused silica presents physical-chemical characteristics compatible with UV-Vis detection, as it is almost transparent to the radiation in this part of the spectrum. Therefore, the detector most frequently used is the UV-Vis, followed by laser-induced fluorescence (LIF) detectors and mass spectrometers. However, other detection systems such as those based on amperometry, conductivity, and light-emitting diodes are also used.

3 THEORY AND PRINCIPLES OF CE The capillary inner wall contains silanol groups that get ionized gaining negative charge in contact with the separation buffer (as shown in Fig. 2). The ionization degree is basically controlled by the separation buffer pH (negative

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- -+-+-+- +- +- +- +- +- +- +- +- +- +- +- +- +- +

–

Anode

µeo

0

µeo

+

µe

µ e+

µ e–

_ Cathode

+ + + + + + + + + + + + + + + +

------------------FIG. 2 Electrophoretic separation by free-solution capillary electrophoresis (FSCE) of three types of substances: with positive electric charge (+), with negative electric charge (), and neutral substances (0).

charges appear in aqueous solutions with pH over 3–4). The wall, negatively charged, attracts the cations from the buffer creating an electrical double layer. This double layer has two zones; one of them fixed next to the capillary wall, where the interactions between the negatively charged silanol groups and the positive ions of the buffer are so strong that they compensate for the thermal agitation; and another zone, further away from the wall, named diffuse. Under the action of the electric field, the positive charges of the diffuse zone move to the cathode and drag with them the associated solvatation water. The result is a global movement of the buffer inside the capillary toward the cathode and it is defined by the electroosmotic mobility, μeo: μeo ¼

εζ η

where ε is the buffer dielectric constant, η is the buffer viscosity, and ζ (zeta potential) can be approximately defined as the potential generated between the negative charge excess at the capillary surface and the positive charge excess at the double layer. This last factor will determine, among other parameters, the electroosmotic flow magnitude. This electroosmotic flow will move all the substances in the interior of the capillary at the same speed because it is a system property, that is to say, it will not introduce selectivity and, therefore, it will not permit the separation of the substances. One of the most important characteristics of this electroosmotic flow is that the flow profile is nearly flat inside the capillary and it provides high separation efficiency compared with the typical parabolic profile of a fluid moving under hydrodynamic forces as in highperformance liquid chromatography (HPLC). Moreover, under an electric field, the charged substances undergo an additional electromigration process inside the capillary, in which each charged analyte tends to move to its opposite pole. Thus, ions experience two opposite forces: one of them due to the electric field (electroosmosis plus/minus electromigration) and the other due to the friction. Using Stokes approximation, where

666 Modern Techniques for Food Authentication

the particle is considered as a rigid sphere, the friction force (Fr) for a substance in any media is given by the equation Fr ¼ 6πηrpve, where rp is the particle radii, η is the media viscosity, and ve is the particle velocity. On the other hand, the charged particle in an electric field undergoes an electric force: Fe ¼ qE, where q is the charge of the particle under the electric field E. The electric field is the result of dividing the applied voltage by the total capillary length. Both forces become equal Fr ¼ Fe, thus, the particle takes a linear uniform movement, where the velocity has the following expression: νe ¼

q E 6πηrp

Being defined the electrophoretic mobility, μe, equal to: μe ¼

q 6πηrp

The electrophoretic mobility is the parameter that controls the selectivity of the separation system through the relation q/rp in the form of free-solution capillary electrophoresis (FSCE), which is the most common mode of CE. As it will be seen below, there can exist other parameters that depending on the mode of CE used can control this selectivity, as for example, the hydrophobicity, the isoelectric point, etc. The relation q/rp is directly related with the ratio charge/volume of the substances. That is to say, for a group of substances with the same amount of electrical charge, the substances with a greater molecular size will have a relation q/rp lower and their electrophoretic mobility μe will be minor, being able to be separated from those substances of a smaller size and, therefore, with higher electrophoretic mobility. In a fused-silica capillary, usually both electrophoretic and electroosmotic migration are simultaneous and can add or subtract depending on the electric charge of the substances as shown in Fig. 2. Frequently the electroosmotic mobility due to the capillary wall is higher than the electrophoretic mobility of the analytes. Thus, the final velocity that the substances are going to adopt inside the capillary will be the addition or subtraction (according to whether they go in the same or in the opposite direction) of these factors: ν ¼ ðμeo  μe ÞE And the specific migration time of a charged substance will then be given by the expression: tm ¼

l ðμeo  μe ÞE

where l is the capillary length from the injection to the detection point.

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MODES OF CE

There are different modes of CE, mainly based on both the nature of the separation media introduced in the capillary, and the characteristics of the analytes to be separated. Interestingly, the instrumentation is practically the same for all of them. Although some others modes, including capillary isotachophoresis (Pospichal et al., 1989), have been described for CE (Erny and Cifuentes, 2006; Venema et al., 1998), they are seldomly used in food authenticity. In the following sections, a short description of the main existing and more frequently used CE modes in this area is given.

4.1 Free-Solution Capillary Electrophoresis FSCE is nowadays the most frequently CE mode used (Jorgenson and Lukacs, 1983). The capillary is filled with a plain buffer solution, allowing the simultaneous separation of positively and negatively charged substances when the magnitudes of the electrophoretic and electroosmotic mobilities are suitable. Following these criteria, compounds with higher positive charge density and smaller radius will migrate the first ones. FSCE presents several limitations (frequently some of them can be overcome by using other different CE modes) that can be summarized as follows: (a) generally, separation of uncharged species or analytes with the same charge to mass ratio [as, e.g., DNA fragments or protein-sodium dodecyl sulfate (SDS) complexes] cannot be accomplished by using FSCE; (b) compounds bearing a high positive electrical charge density can be adsorbed onto the capillary wall (this adsorption will influence negatively the separation process); (c) coefficients of variation for peak areas are in the range from 2% to 5% in real samples analysis; and (d) the sensitivity of the technique does not permit the trace analysis. It is interesting to remark that points (c) and (d) are common for all the CE modes.

4.2 Micellar Electrokinetic Chromatography This CE mode was initially developed to solve the limitation related to the separation of noncharged compounds (Terabe et al., 1984), although it can also be applied to the separation of charged substances. Micellar electrokinetic chromatography (MEKC) involves the addition to the separation buffer of a surfactant at a concentration level at which micelles are formed. These micelles constitute a stable second phase, which in chromatographic terms acts as a pseudo-stationary phase that moves into the capillary, usually SDS micelles are used in MEKC bearing negative charge and, therefore, trending to migrate to the anode. Neutral analytes will interact with the micelles depending on their specific partition coefficient. Fig. 3 shows the separation of three neutral substances with different affinity for the micelles, namely, a

668 Modern Techniques for Food Authentication

P

+

T

N P

Anode µem

N

N

_ Cathode

µeo

FIG. 3 Electrophoretic separation by micellar electrokinetic chromatography (MEKC) of three neutral compounds (T, P, and N) with different hydrophobicities.

compound T that irreversibly interacts with the micelles, a compound P with a medium interaction, and a compound N that does not interact with the micelles. Thus, the migration time of compound T (tm) that interacts irreversibly with the micelles will be the same as that of the micelles. It will depend upon the electroosmotic flow and the electrophoretic mobility of the micelles (μeo and μem). Compound P partially interacts with the micelles and its migration time (tp) will depend as much upon the electrophoretic and electroosmotic mobilities as upon the compound partition coefficient between the aqueous buffer and the micelles. Compound N does not interact with the micelles. As it has no charge, the only driving force to the detector will be the electroosmotic flow. Therefore, it has a migration time (to) corresponding to the electroosmotic mobility (which can be considered as a factor similar to the dead volume in HPLC). The difference between tm and to is the so-called separation window. The compounds to be separated will have migration times within this window and this fact limits the separation power of MEKC. In summary, the mechanism of separation depends on differences in distribution coefficients of the analytes between aqueous and the micellar pseudo-stationary phase.

4.3 Capillary Gel Electrophoresis In this type of CE, the capillary is filled with a buffer solution containing a gel that will act as a molecular sieving. The most important application of this technique is the separation of compounds with the same charge/mass ratio, but with different molecular mass (Cohen and Karger, 1987), as for example, DNA fragments, polysaccharides, SDS-protein complex, or ionic polymers. In capillary gel electrophoresis (CGE), the molecules with smaller molecular size are able to pass through the pores of the molecular sieving formed inside the capillary and migrate first, whereas larger molecules are retarded by the gel and migrate later. The first gels to be used in the latter 1980s were made of cross-linked polyacrylamide covalently linked to the capillary wall. However, they showed many problems related to low reproducibility, resistance, and stability. Nowadays, they have been substituted by the polymeric networks. They are hydrophilic

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noncross-linked polymers that are dissolved in the buffer solution in a concentration usually higher than the so-called entanglement concentration, over which a net is formed that acts as a molecular sieve, although according to Barron et al. (1993), it is not necessary to reach that concentration to obtain the effect of a molecular sieve). Some of the more frequently used polymers are linear polyacrylamide, polyethyleneglycol, polyethylenoxide, polyvinylalcohol, hydroxiethylcellulose, methylcellulose, etc.

4.4 Capillary Isoelectrofocusing The technique capillary isoelectrofocusing (CIEF) has been mostly applied to the separation of peptides and proteins, as shown in the pioneer work published by Hjert
en and Zhu (1985). Usually, a mixture of ampholytes with different pH values is introduced in the capillary together with the sample (the peptides and proteins to be separated). When an electric field is applied, a pH gradient inside the capillary is first established due to the ampholytes, which are distributed from the anode (with low pHs) to the cathode (with high pH values). Peptides or proteins with positive or negative charge, under the influence of the electric field, move through the capillary to the anode or cathode until they reach the zone of the capillary in which the pH of the buffer is the same as their isoelectric point, that is to say, they get a pH value in which the number of their positive and negative charges is the same. At this pH value, analyte migration stops, as its global electrical charge will be zero. When all the compounds have achieved their isoelectric point within the capillary, elution is generally performed by applying a low pressure (keeping on the run voltage) in the anodic end, moving the focused bands toward the detection point. The capillaries used in this mode usually have an internal coating that decreases or eliminates the electroosmotic flow, because that flow would prevent in most cases the formation of the pH gradient.

4.5 Capillary Electrochromatography This type of CE has a great similarity with liquid chromatography. In capillary electrochromatography (CEC), the capillary is filled with silica particles (3–10 μm of diameter and derivatized or not) that act as a stationary phase. The buffer acts as a mobile phase that moves when an electric field is applied. Its velocity is proportional to the electroosmotic flow (i.e., νeo ¼ μeoE). Neutral compounds are carried by the electroosmotic flow and they interact specifically with the stationary phase (in the same way as in HPLC) what originates their separation. As happened with MEKC, CEC mode was mainly developed to separate noncharged compounds in CE (Knox and Grant, 1987). This technique is currently under development with the short life of the packed capillaries as one of its main limitations. These capillaries, apart from being time consuming to

670 Modern Techniques for Food Authentication

prepare and/or expensive, frequently cause the formation of bubbles in the interior as a result of the application of the electric field. This makes the capillaries useless for further applications. Moreover, the employment of CEC to “reallife” samples has still to be proved.

5 APPLICATION OF CE TO FOOD AUTHENTICATION The analytical methodologies available for food quality validation are commonly based on the use of biomarkers and profiling techniques for the characterization of food matrices and identification of adulterants. CE-based methods have demonstrated to be very useful analytical tools for these purposes, due to their abovementioned properties of high separation efficiency and speed, together with the extremely small sample and reagents required. CE has been applied to the analysis of a huge number of food matrices with high incidence of adulteration in the literature search, such as dairy products, meat products, fish and seafood, oils and fats, fruit juice, coffee and tea, alcoholic beverages, spices and extracts, sweeteners cereals, and organic foods among others (D’Orazio et al., 2016; Kvasnicka, 2005). In combination with chemometrics, CE was shown to be a powerful tool for delineating subtle differences among different species, geographical origins, food processing technologies, etc. Some relevant and representative CE approaches applied to the analysis of DNA, proteins, and other food constituents to solve different food authenticity problems are discussed below.

5.1 DNA Analysis Since the introduction of molecular technologies for species identification in the 1980s, the use of DNA-based techniques in food authentication was regarded as a breakthrough in food science. Accurate and sensitive results can be obtained using DNA polymorphism between species as biomarkers for authenticity testing. The high stability of DNA molecules allows the analysis of highly processed food products, as well as trace contaminants, using techniques that allow the rapid amplification of specific DNA sequences, for example, polymerase chain reaction (PCR), which have been fundamental in this field. Thus, most of molecular technologies based on DNA analysis for food authentication depend on the highly specific PCR-based amplification of DNA fragments (Lo and Shaw, 2018). One of the main drawbacks of DNA amplification methods accounts for the necessity of sensitively detecting the presence of the target sequence (amplicon) after the amplification. This final step has been traditionally performed by agarose gel electrophoresis (AGE). Besides the insufficient resolution and sensitivity of AGE, the use of carcinogenic substances and the need to visualize the amplicons were not user-friendly enough for adequate diagnostic setting.

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To overcome such limitations, the combination of CGE with PCR-based methods to successfully amplify and detect DNA sequences with high sensitivity and specificity in an automatic mode, was proposed as an effective solution (Garcı´a-Can˜as et al., 2014). Alternatively, novel DNA-sequencing approaches, known as nextgeneration sequencing (NGS) technologies have transformed genomic science, due to their improved capability for rapidly and inexpensively sequence billions of nucleic acid bases. In this context, CE is a high-throughput separation method still playing an essential role in DNA analysis. For instance, Sanger sequencing via CGE is commonly used to correct for errors in assembling the sequence data, in long repeats of DNA polymer. Thus, CGE is reported as an analytical technique to assist and improve quality control in NGS (Durney et al., 2015). It is worth to notice that in recent years, most of de published CE methods, in combination with the abovementioned molecular techniques for DNA analysis, have been aimed at assessing food authenticity and traceability. The detection and identification of genetically modified organisms (GMOs) in food samples represents an important issue in the food authenticity testing process, considering the legal restriction in terms of approved and unapproved GMOs that can be present in foodstuffs. The benefits of using PCR in combination with CGE to detect GMOs in foods were first reported by Garcı´a-Can˜as et al. (2002), demonstrating that CGE-LIF provided better resolution and a signal/noise ratio improvement of c. 700-fold compared with AGE, and that CGELIF can be a useful tool for the optimization of the multiplex PCR conditions. Following these pioneering works, other PCR amplification-based techniques, such as quantitative competitive PCR (QC-PCR) and multiplex PCR, have also demonstrated great potential when combined with CGE. Holck and Pedersen (2011) developed a multiplex QC-PCR strategy using primers labeled with different fluorescent dyes that in combination with CGE-LIF enables the detection of five genetically modified (GM) maize lines (DAS59122, LY038, MON88017, MIR604, and Event 3272). CGE-LIF was demonstrated to offer a great capability for the accurate and sensitive estimation of transgenic DNA fragments and its respective competitor DNA fragments in QC-PCR reactions. Using two multiplex PCR methods, Basak et al. (2014) developed strategies to specifically detect trace amounts of insect-resistant cotton (MON531) and herbicide-tolerant soybean (GTS40-3-2). Optimal conditions for multiplex reactions were established using primer pairs, in which the forward primer was fluorescently labeled with one of the two fluorophores (6-carboxyfluorescein and hexacloro-6-carboxyfluorescein) used in the study. This strategy allowed the generation of several fluorescent PCR products with similar size, but different fluorescent features, which also provided additional discriminatory information about the amplified DNA sequence when analyzed by CGE equipped with a charge-coupled device detector. Another multiplex PCR method combined with CE was also developed by Mingzhe et al. (2016) to specifically detect four GM rice events. This time, fluorescence multiplex PCR was

672 Modern Techniques for Food Authentication

performed using fluorescence-labeled primers for endogenous, exogenous, and event-specific genes, reaching sensitivity levels as low as a 0.1%. DNA profiling techniques aimed at the analysis of polymorphisms are also used in food authentication. The most popular of these techniques is the restriction fragment length polymorphism (RFLP) analysis which is based on the examination of polymorphic DNA loci characterized by a variable-length restriction fragments. The combination of RFLP analysis with commercial microfluidic CE instruments for the analysis of genetic markers has gained popularity in food authentication. Commercial miniaturized CGE solutions can be found in recent scientific publications, offering many advantages such as simplicity and speed of analysis. For instance, using commercial QIAGEN QIAxcell CGE microfluidic system, Barakat et al. (2014) developed a procedure for detection of pork adulteration in sausages. Authors optimized various porcineand pork-specific PCR systems based on amplifications of mitochondrial D-loop and cytochrome b genes, and used 18S ribosomal gene sequence as internal control for amplification. Then, the specificity of amplified products was assayed using the miniaturized CGE system. The method was robust and suitable for detecting 0.1% pork adulteration in raw and cooked sausages. Another method based on PCR-RFLP and QIAxcell separation technology was developed for the discrimination of oils from 17 Turkish olive cultivars (Uncu et al., 2015). To discriminate the varietal origin of the samples, five PCR-RFLP systems were necessary. In the same line, Spaniolas et al. (2014) and Bazakos et al. (2016) studied the potential of a small number of polymorphisms (SNPs) to evaluate the authentication and traceability of extra-virgin olive oil. Specific SNPs were used as PCR analytical targets and a PCR-RFLP CE approach was used to discriminate several olive oils of Mediterranean origin from three different countries, Greece, Tunisia, and Lebanon. CE in combination with the DNA extraction protocol resulted in lower limits of detection (LODs) compared with previous works. Apart from RFLP analysis, other DNA profiling methods have been used in combination with CGE for ensuring authenticity of the food supply. This is the case for both, random amplified polymorphic DNA (RAPD) and microsatellite marker analysis. Microsatellites, which are short tandemly repetitive DNA sequences, are considered good genetic markers for traceability because of their abundance and high polymorphism. The benefit of using CGE-LIF to detect the amplified DNA products (fingerprint) is to ensure the detection of lowconcentration products and thereby to ensure the integrity of the genomic fingerprint. Thus, Rolli et al. (2014) proposed a method to tackle an authenticity issues related with European perch (Perca fluviatilis), an economically relevant freshwater species in Europe, using microsatellites as DNA markers. Authors combined specific methods for microsatellite markers generation with fluorescent labeling and CGE separation using a conventional CE apparatus coupled to a laser module to increase sensitivity and precision of genotyping. Using this approach, three polymorphic microsatellites and their combinations allowed

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correctly assigning or excluding 60 out of 62 Swiss perch samples into their origin population. Microbial fingerprint has become an emerging authenticity indicator, based on a key assumption that the microbial communities found on food are specific to the geographic area of origin. The occurrence and composition of microbiota in nonprocessed foods like fresh fruits and fish and in fermented foodstuffs such as wine and yoghurt, depends mainly on the cultivation environment of food including soil ecology, insects, and disease agents. PCR-denaturing gradient gel electrophoresis technique (DGGE) is usually used for microbial fingerprinting (El Sheikha and Montet, 2016; Kaur et al., 2015). It is ultra-sensitive since it can distinguish two DNA molecules that differ by as little as a single base. This technique is reliable, reproducible, rapid, inexpensive, and can analyze samples in a high-throughput fashion. The PCR-DGGE has the advantage that separation does not depend on the size of the fragment, but the melting behavior of the PCR product (Danezis et al., 2016). Exposure to food allergens poses significant health risks to allergic consumers, affecting a steadily increasing number of people. Therefore, as part of the authenticity and traceability testing process of food products, the fast and effective detection of potential allergens is essential for food manufacturers to ensure accurate labeling of their allergen-free products. In this regard, Cheng et al. (2015) developed a decaplex PCR assay combined with CE analysis for the simultaneous detection of 10 common food allergens from hazelnut, pistachio, oat, sesame, peanut, cashew, barley, wheat, soybean, and pecan. This method showed LOD between 2 and 20 copies of haploid genome, and the relative LOD was as low as 0.005% (w/w), demonstrating that multiplex-PCR (MPCR) assay is a suitable procedure for routine simultaneous detection of multiple food allergens. Another multiplex alternative was proposed by Maria Lopez-Calleja et al. (2017) who developed a method based on multiplex ligation-dependent probe amplification (MLPA) for simultaneous detection of five food allergens including sunflower, poppy, flaxseed, sesame, and soy in processed food. Ligated MLPA half-probes were amplified by PCR and the resulting amplicons were detected by CE. MLPA is a high-throughput technique, which allows the detection of multiple DNA sequences with more flexibility. The easy validation of MLPA is another advantage compared with MPCR.

5.2 Analysis of Proteins Proteins determination in food samples has demonstrated to be of relevance to carry out the differentiation and identification of species and variety in raw materials. Although the protein composition of raw materials was usually examined by polyacrylamide gel electrophoresis slabs, nowadays the use of analytical techniques based on CE improves sensitivity and accuracy in the protein analysis. One of the main drawbacks of CE in this field is that the protein separation using fused-silica capillaries is strongly hampered by their adsorption

674 Modern Techniques for Food Authentication

onto the capillary wall which is generally due to electrostatic interactions between positively charged residues of the proteins and negatively charged silanol groups. This phenomenon is one of the main reasons for observed efficiency loss, poor reproducibility in migration times, and low protein recovery rates. However, it is possible to employ different strategies to reduce these interactions. Among them are (i) the use of highly alkaline or acidic buffers, (ii) the addition of substances to the separation buffer able to shield the negative charges of the capillary wall (polymers, high salt concentrations), (iii) the use of physically adsorbed coatings of the capillary wall, and (iv) to perform chemical modification of the silica surface. Some of these approaches have been used for protein profiling by CE in a great variety of foods (Acunha ´ lvarez et al., 2018; Garcı´a-Can˜as et al., 2014). et al., 2016a; A CE strategies have demonstrated that protein patterns are a powerful tool to discriminate food samples according to their origin. For instance, using enzyme-assisted extraction prior to CE analysis, and combining the protein profiles established by both CZE-UV (Vergara-Barbera´n et al., 2014b) and CGE (Vergara-Barbera´n et al., 2014a) with linear discriminant analysis was possible to classify correctly olive leaves and pulps coming from different cultivars with an excellent resolution among all the categories. Lately, Vergara-Barberan et al. (2017) have also employed the same CGE methodology as tool to discriminate between cultivars of citrus (orange and tangerine) peel and pulp. Following the same workflow, that is, enzyme-assisted extraction, CGE analysis and linear discriminant analysis, the citrus fruit samples were differentiated, demonstrating that their protein profiles were distinctive of each cultivar (see Fig. 4). The identification and quantification of the mayor whey and casein proteins in dromedary camel milk can be performed by CE, which has several advantages compared with the SDS-polyacrylamide gel electrophoresis (PAGE) method including the simplicity, good resolution, and low costs (Omar et al., 2016). Quantitative differences of the protein composition of camel and bovine milk differs were obtained which can be used for the evaluation of the quality of dairy products made from camel milk and provide new opportunities to assess milk authenticity. A common fraud in dairy industries is the contamination of ewe milk with cow milk. To achieve a rapid recognition of ewe milk adulteration with cow milk, Trimboli et al. (2017) employed a routine CE method for human blood and urine proteins, which fulfilled the separation of skimmed milk proteins in alkaline buffer, to quantify ewe milk in ovine/bovine milk mixtures. Different CE protein profiles were obtained for ovine and bovine milk with a specific ewe peak. With this methodology, it was possible to discriminate up to 95% ewe milk and to detect as low as 5% of cow milk in a mixture with ovine milk.

5.3 Analysis of Chiral Compounds The relevance of enantioselective separations for identifying adulterated foods and beverages was already established by Armstrong et al. (1990). The first

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FIG. 4 (A) CGE-laser-induced fluorescence (LIF) electropherograms corresponding to the protein profile of peels and pulps extracts of citrus fruits (enzyme peaks were labeled with an asterisk), and score plots on an oblique plane of the three-dimensional (3D) space defined by the three first discriminant functions of the linear discriminant analysis model constructed to classify citrus pulp (B) and citrus peels samples (C) according to their cultivar. (Redrawn from Vergara-Barberan, M., Mompo-Rosello, O., Jesus Lerma-Garcia, M., Manuel Herrero-Martinez, J., Francisco Simo-Alfonso, E., 2017. Enzyme-assisted extraction of proteins from Citrus fruits and prediction of their cultivar using protein profiles obtained by capillary gel electrophoresis. Food Control 72, 14–19.)

676 Modern Techniques for Food Authentication

enantiomeric separation by CE was published by Gassmann et al. (1985), 30 years ago. From them till now, the use of CE in chiral separations has continuously grown until be established as one of the most employed and powerful techniques for analytical enantioseparations. The use of CE for enantiomer separations provides fast and efficient separations in this type of analysis. In addition, the availability of many chiral selectors and the minimum consumption of such compounds during a CE run have to be considered as an additional advantage of capillary electromigration methods. The development of chiral analytical methodologies for the determination of different compounds such as α-hydroxy acids, amino acids, or phenolic compounds provides interesting tools to fight against food adulteration. Different strategies based on the use of ligand exchange CE-UV have been developed to analyze α-hydroxy acids in juices. One of the most relevant parameters used to distinguish authentic and adulterated fruit juices is the ratio of citric acid to D-isocitric acid. For this reason, D- and L-isocitric acid enantiomers were separated using D-quinic acid as chiral selector and Ni(II) as central ion system. Using this procedure, the contents of DL-isocitric and citric acids were successfully estimated in different fruit juices being in agreement with the fruit juice industry Code of Practice (Kodama et al., 2010). The enantiomeric determination of other α-hydroxy acids, such as DL-malic acid or DL-tartaric acid can also be used as indicators of adulteration. For instance, the analysis of these organic acids in different juices samples by using a ligand exchange CE-UV with D-quinic acid as chiral selector and a dual central metal ion system enabled to identify the addition of racemic malic acid to the grape juice (L-malic acid is one of the principal organic acid in fruits juices however, no D-malic acid should be present) (Kodama et al., 2013). The ligand exchange principle using L-histidine as chiral ligand and copper (II) as central ion in an open tubular CEC methodology was also useful for the enantiomeric determination of malic acid in apple juice (Aydogan et al., 2015). On the other hand, the combination of copper (II)/D-quinic acid system along with the use of hexadecyltrimethylammonium as electroosmotic flow reversal agent was applied to the quantitative analysis of DL-malic acid or DL-tartaric acid in wine samples. By using this methodology was possible to detect D-malic acid in different wines in a broad range of concentrations and D-tartaric acid in some samples. The data obtained are of great relevance for the quality control of the analyzed wine, since while the addition of DL-malic acid for wine acidification is legal, the use of DL-tartaric acid is not allowed by the International Organization of Vine and Wine (Kamencev et al., 2016). MEKC with LIF detection has been also employed for evaluate food adulteration through the analysis of derivatized D- and L-amino acids in juices. In this case, representative groups of D- and L-amino acids were derivatized with fluorescein isothiocianate (FITC) to enhance the sensitivity. This derivatization also facilitates the chiral separation of amino acids, since inclusion and interaction with the chiral selector becomes more discriminating. Using sodium

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dodecylbenzene sulfonate as surfactant and β-CD as chiral selector, a mixture of amino acids (L-Arg, D-Leu, L-Trp, L-Leu, L-Pro, D-Pro, L-Asn, L-Ser, L-Ala, L-Glu, and L-Asp) was analyzed in pomegranate juices. The comparison of the amino acid profile of pomegranate and apple juices enabled to propose L-Asn as a marker for the adulteration of pomegranate juices with apple juices (Tezcan et al., 2013). The study of catechines epimerization by CE methodologies has also demonstrated to be useful to evaluate food authenticity. In this regards, Kofink et al. (2007) demonstrated the potential of an electrokinetic chromatography-UV method based on the use of hydroxypropyl-γ-cyclodextrin as chiral selector to investigate the authenticity of guarana´ through the analysis of catechin and epicatechin enantiomers.

5.4 Analysis of Other Compounds Besides the aforementioned molecular techniques based on DNA, proteins, and amino acid analysis, other CE methods have been described in literature for the analysis of a large number of food components, such as saccharides, inorganic cations (minerals), organic acids, phenolic compounds, etc. (Acunha et al., ´ lvarez et al., 2018; Castro-Puyana et al., 2012; Garcı´a-Can˜as and 2016a; A Cifuentes, 2008; Garcı´a-Can˜as et al., 2014; Herrero et al., 2010). These analytes are of considerable significance in food authenticity testing, and the most representative advances in CE for their analysis are discussed below. CE can be used to analyze both the organic acids and mineral contents in order to evaluate, for example, the authenticity of wine samples. To illustrate this point, Zeravik et al. (2016) employed CE-UV for the determination of the organic acids composition and quantification of 31 wine samples and for the classification of 38 samples of commercial wine from four wine-producing provinces of Argentina according to the grape variety applying the Tucker3 algorithm. Besides, Vaughan et al. (2016) evaluated the changes in organic acids levels in coffee seeds as function of different fermentation treatments. In order to characterize Anatolian monofloral and honeydew honeys according to their mineral, vitamin B2, total phenolic contents, and antioxidant activities, Kaygusuz et al. (2016) used a CE method coupled with LIF detector to determine vitamin B2 contents. The results showed that riboflavin concentrations of heather honey samples are considerable higher than vitamin B2 contents of other honeys. A similar method like the one previously described was used for the determination of the vitamin B2 level in saffron developed by Hashemi and Erim (2016). The CE system was equipped with a LIF detector and a fused-silica capillary. Five commercial samples of saffron, three from Iran and two from Spain, were analyzed. Comparing the riboflavin contents of these samples with the reported riboflavin contents of other food sources in the literature, it could be established that saffron is one of the most riboflavin-rich foods.

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CE has also proven to be a powerful separation technique for the separation and analysis from small mono- and disaccharides to complex oligo and polysaccharides. Since maple syrup is prone to adulteration with less expensive sugars (i.e., corn syrup), saccharide content from maple syrup was investigated through CE-UV with borate complexation (Taga and Kodama, 2012). Among all saccharides, it was observed that the main components of maple syrup and maple sugar were sucrose, glucose, and mannose. Owing to economic value of fruit juices, they are also prone to adulteration by dilution with water and addition of sugars or of pulp wash, or even through mixture with cheaper fruit juices. In this regard, a capillary zone electrophoresis (CZE)-UV method was developed for the differentiation of juices and blends based on their saccharides content (NavarroPascual-Ahuir et al., 2015). More than 50 samples including juices from apple, grape, mandarin, orange, pineapple, and nectar from orange, pineapple as well as multifruit juices were evaluated. Juices and blends could be correctly classified based on their saccharides content and adulteration could be predicted by means of linear discriminant analysis and multiple linear regression. The study of the phenolic compound profiles by CE techniques was shown to be very useful for the classification of different natural samples. Polyphenolic profiling has been applied for the authentication of fruit products combining CZE and liquid chromatography (LC) techniques. Samples under study included fruits (cranberry, blueberry, grapes, and raisins), fruit-based products (grape juice and cranberry juice), and commercial cranberry-based products (Navarro et al., 2014). After data analysis using Matlab, the resultant principal components analysis (PCA) showed a clear clustering according to the fruit origin as can be observed in Fig. 5. Moreover, one sample was suspected to be an adulteration using the proposed LC and CE methods. The high-resolution power of CE using a fused-silica capillary, allowed the fingerprint study of the extracted compounds of 10 Mentha herbal samples and 20 peppermint teas (Roblova et al., 2016). The different phenolic profiles of each sample, together with spectrophotometric methods for the determination of the total phenolic compounds and the antioxidant capacity of each extract allowed distinguishing the Mentha and peppermint tea samples by a PCA model according to their potential protective antioxidant effect. Another CE method for the classification and the study of the impact of the aging length, aging process, and mash bill of different Irish whiskies on their phenolic profile has been reported (White et al., 2017). In this case, a field amplified sample stacking (FASS) preconcentration approach was employed to increase the sensitivity of the analytes of interest. This method is very useful for samples of low conductivity such as the whiskey matrices. Results showed that the length of aging in Irish whiskies positively affect the concentration of phenolic acids; whiskey aging in sherry casks produced a final product with a greater number of phenolic compounds types; and finally, the phenolic profile of single pot still whiskies resulted in a rich concentration of phenolic aldehydes and a diversity of phenolic acids. Fatty acids (FA) are a group of lipids which turn out to be of great importance as biomarkers for food authentication purposed. A CZE-UV method was

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FIG. 5 (A) PCA result (PC3 vs PC4 score plot) using electrophoretic polyphenolic profiles for fruits and juice samples. (B) PCA results (PC4 vs PC5 score plot) using electrophoretic polyphenolic profiles for all samples except pharmaceutical capsules. (C) Loading plot using peak signal areas. (D) Electropherogram obtained for a cranberry commercial capsule. Discriminant peak signals are indicated with an arrow. (E) PCA result (PC3 vs PC4 score plot) using eight discriminant peak signal areas. (Adapted with permission from Navarro, M., Nu´n˜ez, O., Saurina, J., Herna´ndez-Cassou, S., Puignou, L., 2014. Characterization of fruit products by capillary zone electrophoresis and liquid chromatography using the compositional profiles of polyphenols: application to authentication of natural extracts. J. Agric. Food Chem. 62, 1038–1046. Copyright 2018 American Chemical Society.)

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Scores on PC 4 (0.46%)

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developed to detect adulteration in milk samples by FAs analysis (de Oliveira Mendes et al., 2016). Addition of whey on milk is known to be one of the main dairy products adulterations issues. In this regard, the six most important FAs present in milk and whey were selected in this work as the six FAs that could be the most significant to detect the adulteration. The proposed method allowed the detection and quantification of whey addition in a range of 4%–20% of adulteration, resulting to be a good method for quality control of milk samples.

5.5 CE Microchip Technology in Food Authentication Microchip electrophoresis (MCE) is considered as a very attractive tool for detecting food fraud and for rapid analysis of a wide variety of hazardous substances in food, representing a helpful alternative to ensure food safety control. Similarly to CE, MCE has several advantages such as the high efficiency or the low consumption of reagent and sample. Besides these benefits, MCE also presents a great potential for miniaturization and the possibility of carrying out fast and real-time analysis, which might be also performed in situ. However, despite these interesting analytical advantages, the use of this methodology is still limited, mainly due to the high cost of glass or silica microchips, and also due to the difficulties that the thinner microchannels normally used in MCE pose to the ´ lvarez et al., 2018) analysis (A MCE has been applied to wine differentiation and authentication, analyzing organic compounds such as polyphenols, organic acids, aldehydes, sugars, alcohols, or neuroactive amines, which may provide relevant information about the botanical or cultivar origin, vintage, and quality of different wine samples (Gomez and Silva, 2016). A powerful analytical platform for the analysis of ionic species in beverages was developed by Rezende et al. (2016), coupling MCE to contactless conductivity detection (C4D) for the analysis of ionic species in beverages, for example, whiskey samples. For the study of the authenticity of seized whiskey that could probably be adulterated by the addition of tap water, the presence of anionic species in the seized samples was monitored and compared with the original samples. Another MEC method was used for the analysis of high abundance fish muscle proteins, combining water-based protein extraction with a software algorithm technique for data analysis. In this way, Walker et al. (2017) demonstrated that protein profiles can be applied to distinguish uncooked seafood products containing catfish, snapper, grouper, and other commercially important species from commonly substituted species with a high level of confidence.

6 FUTURE OUTLOOKS Many of the points indicated as future developments in the previous version of this chapter are still in progress nowadays. For example, despite the multiple applications of CE in food authenticity, there are still some problems that have to be addressed before it can be considered as a mature and routine technique in

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this area. Hence, although the volumes of sample usually consumed per analysis in CE are a few nanoliters, the sensitivity in terms of concentration is not very high, which precludes the use of CE for determination of trace compounds. To enhance the sensitivity different strategies have been applied, for example, by using sample preconcentration, stacking procedures, more sensitive detection systems (such as LIF, amperometric detector), etc. Also, in order to improve both sensitivity and mainly selectivity, CE can be interfaced with other techniques such as electrospray mass spectrometry (MS) to bring about a very powerful hyphenated technique (Acunha et al., 2016b). On-line coupling of CE with electrospray-MS may solve the identification problems associated with unknown compounds detected in the complex food matrices usually analyzed. Moreover, the application of CE-MS to food analysis including food authentication is an important and practically unexplored working field. The same ´ lvarez applies to the new discipline of foodomics (Herrero et al., 2012; A et al., 2018) that is expected to play a crucial role in food authenticity investigations in combination with the use of CE techniques.

7

CONCLUSIONS

Despite some problems that have to be addressed in CE methodologies before CE can be considered as a mature and routine technique in food science and technology, in the last years CE has demonstrated to be a real alternative to other chromatographic methods for the assessment of food authenticity. In fact, the inherent characteristics of CE (high analysis speed, high separation efficiency, great variety of applications, and reduced sample and solvents consumption) have contributed to make it an ideal option to face the determination of different compounds such as DNA fragments, proteins, chiral compounds, polyphenols, FA, or carbohydrates for solving many analytical problems related to food authenticity. Instrumental and methodological advances of CE are in continuous development to improve mainly its sensitivity and reproducibility, which allow expecting that the number of applications of CE in food authentication will continue to grow in the future.

ACKNOWLEDGMENTS The present work was supported by the projects AGL2014-53609-P (Ministerio de Economı´a y Competitividad, Spain). G.A.-R. would like to acknowledge MINECO for a Juan de La Cierva-Formacio´n postdoctoral grant (FJCI-2015-25504). M.C.P. also thanks MINECO for her “Ramo´n y Cajal” research contract (RYC-2013-12688).

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682 Modern Techniques for Food Authentication Acunha, T., Simo, C., Ibanez, C., Gallardo, A., Cifuentes, A., 2016b. Anionic metabolite profiling by capillary electrophoresis-mass spectrometry using a noncovalent polymeric coating. Orange juice and wine as case studies. J. Chromatogr. A 1428, 326–335. ´ lvarez, G., Montero, L., Llorens, L., Castro-Puyana, M., Cifuentes, A., 2018. Recent advances in A the application of capillary electromigration methods for food analysis and Foodomics. Electrophoresis 39, 136–159. Armstrong, D.W., Chang, C.D., Li, W.Y., 1990. Relevance of enantiomeric separations in food and beverage analyses. J. Agric. Food Chem. 38, 1674–1677. Aydogan, C., Karakoc, V., Denizli, A., 2015. Chiral ligand-exchange separation and determination of malic acid enantiomers in apple juice by open-tubular capillary electrochromatography. Food Chem. 187, 130–134. Barakat, H., El-Garhy, H.A.S., Moustafa, M.M.A., 2014. Detection of pork adulteration in processed meat by species-specific PCR-QIAxcel procedure based on D-loop and cytb genes. Appl. Microbiol. Biotechnol. 98, 9805–9816. Barron, A.E., Soane, D.S., Blanch, H.W., 1993. Capillary electrophoresis of DNA in uncross-linked polymer solutions. J. Chromatogr. A 652, 3–16. Basak, S., Ehtesham, N.Z., Sesikeran, B., Ghosh, S., 2014. Detection and identification of transgenic elements by fluorescent-PCR-based capillary gel electrophoresis in genetically modified cotton and soybean. J. AOAC Int. 97, 159–165. Bazakos, C., Khanfir, E., Aoun, M., Spano, T., El Zein, Z., Chalak, L., El Riachy, M., AbouSleymane, G., Ben Ali, S., Kammoun, N.G., Kalaitzis, P., 2016. The potential of SNP-based PCR-RFLP capillary electrophoresis analysis to authenticate and detect admixtures of Mediterranean olive oils. Electrophoresis 37, 1881–1890. Bol’shakova, D.S., Amelin, V.G., 2016. Determination of pesticides in environmental materials and food products by capillary electrophoresis. J. Anal. Chem. 71, 965–1013. Castro-Puyana, M., Garcı´a-Can˜as, V., Simo´, C., Cifuentes, A., 2012. Recent advances in the application of capillary electromigration methods for food analysis and Foodomics. Electrophoresis 33, 147–167. Cheng, F., Wu, J., Zhang, J., Pan, A., Quan, S., Zhang, D., Kim, H., Li, X., Zhou, S., Yang, L., 2015. Development and inter-laboratory transfer of a decaplex polymerase chain reaction assay combined with capillary electrophoresis for the simultaneous detection of ten food allergens. Food Chem. 199, 799–808. Cifuentes, A., 2006. Recent advances in the application of capillary electromigration methods for food analysis. Electrophoresis 27, 283–303. Cohen, A.S., Karger, B.L., 1987. High-performance sodium dodecyl sulfate polyacrylamide gel capillary electrophoresis of peptides and proteins. J. Chromatogr. 397, 409–417. D’Orazio, G., Asensio-Ramos, M., Fanali, C., Herna´ndez-Borges, J., Fanali, S., 2016. Capillary electrochromatography in food analysis. TrAC Trends Anal. Chem. 82, 250–267. Danezis, G.P., Tsagkaris, A.S., Brusic, V., Georgiou, C.A., 2016. Food authentication: State of the art and prospects. Curr. Opin. Food Sci. 10, 22–31. de Oliveira, M.A.L., Porto, B.L.S., de A. Bastos, C., Sabarense, C.M., Vaz, F.A.S., Neves, L.N.O., Duarte, L.M., da S. Campos, N., Chellini, P.R., da Silva, P.H.F., de Sousa, R.A., Marques, R., Sato, R.T., Grazul, R.M., Lisboa, T.P., de O. Mendes, T., Rios, V.C., 2016. Analysis of amino acids, proteins, carbohydrates and lipids in food by capillary electromigration methods: a review. Anal. Methods 8, 3649–3680. de Oliveira Mendes, T., Porto, B.L.S., Bell, M.J.V., Perrone, I´.T., de Oliveira, M.A.L., 2016. Capillary zone electrophoresis for fatty acids with chemometrics for the determination of milk adulteration by whey addition. Food Chem. 213, 647–653.

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